The general objective of the proposed research is to contribute to an understanding of the mechanisms involved in the generation of chromosomal aneuploidy and in the expression of subsequent abnormal phenotypes. The proposed experiments take advantage of experimental approaches available in S. cerevisiae and rely on vectors and methods developed in our laboratory for the construction and manipulation of Yeast Artificial Chromosomes (YACs). The specific experimental aims are: 1) To further develop methodologies for YAC cloning, manipulation, and transfer. Methodologies to increase the frequency of monochromosomal transfer of YACs during yeast/mammalian cell fusion will be developed using karyogamy deficient yeast donor strains. A novel "recombinational cloning" method will be further developed and used to clone a functional C. albicans centromere DNA sequence. The effect of centromere DNA sequences on the recombination mechanism of chromosome fragmentation will be analyzed in detail. 2) To analyze determinants necessary for Meiosis I chromosome disjunction. Using a 2-YAC meiosis segregational system developed in our laboratory, cis- and trans-acting determinants will be identified and characterized. The molecular basis of defects causing increased levels of precocious sister chromatid separation, Meiosis I nondisjunction, and Meiosis II missegregation events will be studied. 3) To analyze specific aneuploidies in yeast. The phenotypic effects of chromosome II LYS2-distal monosomy (chromosome transmission defect) will be characterize and the corresponding gene(s) identified, cloned, and characterized. 4) To analyze human homologs of genes required for chromosome disjunction fidelity. Genes will be identified by functional complementation of yeast mutants or by evolutionary conservation of amino acid sequenced and their roles in mammalian cells determined. An understanding of the mechanisms that lead to chromosal aneuploidy and the factors that contribute to the phenotypic consequences associated with specific aneuploidies are major challenges in human biology. The development of methodologies for manipulating chromosomes (including YACs) in yeast will facilitate the analysis of those segments of mammalian DNA leading to an aneuploid phenotype. Moreover, analysis of mechanisms of aneuploidy in yeast will be important to a basic understanding of these processes in general.